ABSTRACT A safe and effective vaccine providing protection from many subtypes of influenza would considerably improve public health and pandemic preparedness. Multiple potentially pandemic viruses continue to circulate and evolve in the environment causing public health concern. Preparation of vaccines for each subtype using current technologies is not cost-effective and can raise safety and biosecurity concerns if highly pathogenic strains are used. Recombinant virus-like particles (VLPs) represent an intrinsically safe vaccination approach. Influenza VLPs contain viral hemagglutinin (HA), neuraminidase (NA) and matrix or gag proteins, which self-assemble into VLPs in cell culture. VLPs morphologically and antigenically resemble influenza virions except they are non-infectious. Recombinant VLPs have advantages in safety, efficacy, and manufacturing and they circumvent problems like slow virus growth, unpredictable yields, and virus mutations during egg adaptation. In preliminary studies, we described a novel multi-subtype VLP design that co-localizes multiple HA subtypes within the same VLP particle [1, 2]. VLP that contained HA proteins from four distinct avian influenza subtypes H5, H7, H9, and H10 proteins induced specific immune responses against all four subtypes. Therefore, multi-subtype VLP design suggests the potential for a broadly protective vaccine that provides specific immunity against multiple influenza viruses of pandemic concern. In this Phase I SBIR application, we propose feasibility study of a novel multivalent vaccine containing HA molecules from all seven zoonotic pandemic threat subtypes known to infect humans including these of avian and swine origin. In Sp. Aim 1, VLP vaccines will be prepared in the mono- and multi-subtype formats and optimized for expression of H1, H2, H3, H5, H7, H9, and H10 antigens. VLPs will be expressed using a baculovirus expression system and their structural, antigenic, and biochemical characteristics will be evaluated. The content and the potency of each HA subtype will be measured to determine optimal formulation of VLPs. In Sp. Aim 2, safety, immunogenicity and efficacy of the best VLP formulation will be assessed in experimental ferret model in collaboration with the Centers for Disease Control and Prevention (CDC). Ferrets will be vaccinated with the optimized multivalent vaccine from Sp. Aim 1. Immune responses to the expressed avian and swine HA subtypes will be determined including hemagglutination inhibition (HI) and virus neutralization (VN) titers. In addition, antibodies to NA, as well as IFN? responses after VLP immunization will also be determined. Vaccine efficacy will be evaluated using challenge with at least two homologous viruses. Additional homologous and heterologous challenge experiments are planned for the follow-up Phase II SBIR if approved by the Agency. If successful, this high-risk, high-reward approach can potentially result in a novel emergency vaccine protecting against multiple potentially pandemic viruses known to infect humans.